Isolation of tumor cells using size and deformation

The isolation and analysis of circulating tumor cells (CTCs) from blood are the subject of intense research. Although tests to detect metastasis on a molecular level are available, progress has been hampered by a lack of tumor-specific markers and predictable DNA abnormalities. The main challenge in this endeavor is the small number of available cells of interest, 1–2 per mL in whole blood. We have designed a micromachined device to fractionate whole blood using physical means to enrich for and/or isolate rare cells from peripheral circulation. It has arrays of four successively narrower channels, each consisting of a two-dimensional array of columns. Current devices have channels ranging in width from 20 to 5 μm, and in depth from 20 to 5 μm. Several optimizations resulting in the fabrication of a total of 10 derivative devices have been carried out; only two types are used in this study. Both have increasingly narrower gap widths between the columns along the flow axis with 20, 15, 10, and 5 μm spacing all on one device. The first 20 μm wide segment disperses the cell suspension and creates an evenly distributed flow over the entire device, whereas the others were designed to retain increasingly smaller cells. The channel depth is constant across the entire device, the first type was 10 μm deep and the second type is 20 μm deep. When cells from each of eight tumor cell lines were loaded into the device, all cancerous cells were isolated. In mixing experiments using human whole blood, we were able to fractionate cancer cells without interference from the blood cells. Additionally, either intact cells, or DNA, could be extracted for molecular analysis. The ultimate goal of this work is to characterize the cells on the molecular level to provide non-invasive methods to monitor patients, stage disease, and assess treatment efficacy. Furthermore, this work will use gene expression profiles to gain insights into metastasis.     

Efforts Toward the Direct Experimental Characterization of Enzyme Microenvironments: Tyrosine100 in Dihydrofolate Reductase

State secrets : Site‐specific deuteration and FTIR studies reveal that Tyr100 in dihydrofolate reductase plays an important role in catalysis, with a strong electrostatic coupling occuring between Tyr100 and the charge that develops in the hydride‐transfer transition state (see picture, NADP⁺ purple, Tyr100 green). However, relaying correlated motions that facilitate catalysis from distal sites of the protein to the hydride donor may also be involved.

     

Terahertz spectroscopy techniques for explosives detection

Spectroscopy in the terahertz frequency range has demonstrated unique identification of both pure and military-grade explosives. There is significant potential for wide applications of the technology for nondestructive and nonintrusive detection of explosives and related devices. Terahertz radiation can penetrate most dielectrics, such as clothing materials, plastics, and cardboard. This allows both screening of personnel and through-container screening. We review the capabilities of the technology to detect and identify explosives and highlight some of the critical works in this area.     

Combination of perturbative and variational methods for calculating molecular spectra: calculation of the upsilon=3-5 CH stretch overtone spectrum of CHF3

Highly excited states of the CHF3 molecule belonging to the third, fourth, and fifth Fermi polyad are calculated using a combination of the Van Vleck perturbation theory and a variational treatment. The perturbation theory preconditions the Hamiltonian matrix by transforming away all couplings except those between nearly degenerate states. This transformation is implemented so that eigenvalues can be found with significantly smaller matrices than that which would be needed in the original normal mode representation. Even with preconditioning, at the energies as high as 3-5 quanta in the CH stretch, it is not possible to directly diagonalize the Hamiltonian matrix due to the large basis sets required. Iterative methods, particularly the block-Davidson method, are explored for finding the eigenvalues. The methods are compared and the advantages discussed.     

Surface modification for enhancing antibody binding on polymer-based microfluidic device for enzyme-linked immunosorbent assay

A novel surface treatment method using poly(ethyleneimine) (PEI), an amine-bearing polymer, was developed to enhance antibody binding on the poly(methyl methacrylate) (PMMA) microfluidic immunoassay device. By treating the PMMA surface of the microchannel on the microfluidic device with PEI, 10 times more active antibodies can be bound to the microchannel surface as compared to those without treatment or treated with the small amine-bearing molecule, hexamethylenediamine (HMD). Consequently, PEI surface modification greatly improved the immunoassay performance of the microfluidic device, making it more sensitive and reliable in the detection of IgG. The improvement can be attributed to the spacer effect as well as the functional amine groups provided by the polymeric PEI molecules. Due to the smaller dimensions (140x125 microm) of the microchannel, the time required for antibody diffusion and adsorption onto the microchannel surface was reduced to only several minutes, which was 10 times faster than the similar process carried out in 96-well plates. The microchip also had a wider detection dynamic range, from 5 to 1000 ng/mL, as compared to that of the microtiter plate (from 2 to 100 ng/mL). With the PEI surface modification, PMMA-based microchips can be effectively used for enzyme linked immunosorbent assays (ELISA) with a similar detection limit, but much less reagent consumption and shorter assay time as compared to the conventional 96-well plate.     

Rapid determination of complex mixtures by dual-column gas chromatography with a novel stationary phase combination and spectrometric detection

Fast GC separations of a broad range of analytes are demonstrated using a capillary column coated with a novel immobilized ionic liquid (IIL) stationary phase. Both completely cross-linked and partially cross-linked columns were evaluated, yielding approximately 1600 and approximately 2000 theoretical plates per meter, respectively. Enhanced separation is demonstrated using a dual-column ensemble comprised of an IIL column, a commercially coated Rtx-1 column, and a pneumatic valve connecting the inlet to the junction point between the two columns. Enhanced separation of 20 components, with two sets of co-eluting peaks is shown in approximately 150 s, while sacrificing only a length of time equivalent to the sum of the stop flow pulses, or about 15.5 s. A novel application of a band trajectory model that shows band position as a function of analysis time as analytes move through the column ensemble is employed to determine pulse application times. The model predicts component retention times within a few seconds. Another method of selectivity enhancement of the IIL stationary phase-coated columns is demonstrated using a differential mobility spectrometer (DMS) that provides a second dimension separation based on ion mobility in a high-frequency electrical field. The DMS is able to separate all but one set of co-eluting components from the IIL column. The separation of 13 components found in the headspace above U.S. currency is demonstrated using the IIL column in a dual-column ensemble as well as with the DMS.     

Solvent‐Free Fabrication of polyHIPE Microspheres for Controlled Release of Growth Factors

The growth factor bone morphogenetic protein 2 (BMP‐2) is utilized in surgical procedures to improve bone regeneration; however, current treatments deliver BMP‐2 at amounts greater than 100 000 fold of physiological levels, which increases treatment costs and risk of side effects. Drug‐eluting microcarriers developed to improve these therapies have faced significant commercialization challenges including particle size distributions, solvent removal, low encapsulation efficiency, and bioactivity loss. In this study, a solvent‐free method is presented for fabrication of uniform polyHIPE microspheres for controlled growth factor release. Emulsion templating principles and fluid dynamics were used to fabricate uniform particles with tunable particle size (200–800 μm) and pore size (10–30 μm). The ability to independently tune particle and pore size is expected to provide excellent control of release kinetics. Overall, this solvent‐free method for making porous microspheres displays strong promise for the controlled release of BMP‐2 and other growth factors.

     

Aggregation and supramolecular membrane interactions that influence anion transport in tryptophan-containing synthetic peptides

Self-assembly is a desired property in supramolecular chemistry, but extensive aggregation may be counterproductive. Rigid systems typically have better organization, but are inherently less dynamic. This work shows that ion transport by amphiphilic heptapeptides (synthetic anion transporters or SATs) is affected by aggregation of the monomers in the bulk aqueous phase to which they are added and within the bilayer. Ion transport was assessed for all compounds by assay of Cl(-) release from liposomes. The mechanism of ion transport was confirmed by planar bilayer conductance studies for two compounds at opposite ends of the efficacy scale. Dynamic light scattering, the Langmuir trough, transmission electron microscopy, ion release from liposomes, and planar bilayer conductance studies were used to assess the importance of self-assembly versus aggregation in ion transport. Generally, greater aggregation was has an adverse effect on the transport, although at least dimerization is required for amphiphilic heptapeptides to readily transport Cl(-). Anion transport in these systems was found to be sensitive to changes in the C-terminal portion of the (Gly)(3)Pro(Gly)(3) sequence. Moreover, a significant difference in transport efficacy was apparent when L-Trp was replaced by D-Trp in the same position.     

The development of direct multicolour fluorescence cross-correlation spectroscopy: towards a new tool for tracking complex biomolecular events in real-time

Direct three-colour fluorescence cross-correlation spectroscopy can reveal interactions between three fluorescently labelled biomolecules, giving insight toward the complex events that constitute signal transduction pathways. Here we provide the optical and theoretical basis for this technology and demonstrate its ability to detect specific biological associations between nanoparticle-labelled DNA molecules.     

A self-loading microfluidic device for determining the minimum inhibitory concentration of antibiotics

This article describes a portable microfluidic technology for determining the minimum inhibitory concentration (MIC) of antibiotics against bacteria. The microfluidic platform consists of a set of chambers molded in poly(dimethylsiloxane) (PDMS) that are preloaded with antibiotic, dried, and reversibly sealed to a second layer of PDMS containing channels that connect the chambers. The assembled device is degassed via vacuum prior to its use, and the absorption of gas by PDMS provides the mechanism for actuating and metering the flow of fluid in the microfluidic channels and chambers. During the operation of the device, degas driven flow introduces a suspension of bacterial cells, dissolves the antibiotic, and isolates cells in individual chambers without cross contamination. The growth of bacteria in the chambers in the presence of a pH indicator produces a colorimetric change that can be detected visually using ambient light. Using this device we measured the MIC of vancomycin, tetracycline, and kanamycin against Enterococcus faecalis 1131, Proteus mirabilis HI4320, Klebsiella pneumoniae, and Escherichia coli MG1655 and report values that are comparable to standard liquid broth dilution measurements. The device provides a simple method for MIC determination of individual antibiotics against human pathogens that will have applications for clinical and point-of-care medicine. Importantly, this device is designed around simplicity: it requires a single pipetting step to introduce the sample, no additional components or external equipment for its operation, and provides a straightforward visual measurement of cell growth. As the device introduces a novel approach for filling and isolating dead-end microfluidic chambers that does not require valves and actuators, this technology should find applications in other portable assays and devices.